The present invention generally relates to compositions and methods of detecting molecular interactions, such as protein interaction, in the fields of research, medical diagnostics, and DNA research. Various methods have been developed to study steady state levels, biosynthesis and turnover, mutation and stability, and effects of pharmacological compounds on expression.
Western blots are commonly used to detect proteins. A SDS-PAGE (sodium dodecyl sulfate polyacrylaminde gel electrophoresis) technique is first used to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding, post translational modification and other factors). Following the SDS-PAGE of protein samples and electrophoretic transfer of protein polypeptides from the polyacrylaminde gel to PVDF (polyvinylidene fluoride) or nitrocellulose membranes, polypeptides are identified using protein specific primary antibodies, secondary antibody conjugates of peroxidase or alkaline phosphatase enzymes, and chromogenic or chemiluminescent substrates of peroxidase or alkaline phosphatase enzymes. Protein detection is limited by chromogenic substrates and the read out of results on a western blot is inconvenient, difficult and often not accurate. These difficulties led to the use of chemiluminescent substrates for peroxidase or alkaline phosphatase or other enzymes. This is also a convenient method to detect protein polypeptides on western blotting by using X-ray film.
FIG. 1 illustrates the mechanism of a chemiluminescence reaction. In the chemiluminescence reaction luminol or isoluminol is catalyzed by superoxide anions in a solution with an alkaline pH. Luminol is converted to aminophthalate ions by superoxide anions produced from hydrogen peroxide or sodium perborate by a peroxidase enzyme at alkaline pH. Superoxide anions convert luminol or isoluminol to aminothalate ions which give rise to chemiluminescence light of 430 nm. This light can be recorded by a luminometer or detected on X-ray film. A strong and stable chemiluminescence signal of luminol depends on the alkaline pH of the reaction because strong and stable chemiluminescence is due to the presence of stable aminothalate ions providing enhanced luminescence. Therefore, it would be beneficial to provide a composition and a method for producing the same that has stable aminothalate ions thereby enhancing the chemiluminescence and the detection of proteins and other substances.
The following references that are cited throughout this disclosure are incorporated herein by reference in their entirety to the extent permitted by law. These references merely serve to support the invention and to provide background and context. Applicant reserves the right to challenge the veracity of any statement made therein.
Yakunin, Alexander F. and Hallenbeck, Patrick C. A Luminol/Iodophenol Chemiluminescent Detection System for Western Immunoblots. Analytical Biochemistry 258 (1), 146-149.
Towbin, H. T., Staehelin, T., and Gordon, J. (1979) Electrophoretic Transfer Of Proteins From Polyacrylamide Gels to Nitrocellulose Sheets Procedure and Some Applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.
Vachereau, A. (1989) Luminiscent Immunodetection of Western Blotted Proteins from Coomassie Stained Polyacrylamide Gel. Anal. Biochem. 179, 206-208.
Waheed, A., Zhu, X. L., and Sly, W. S. (1992) Membrane Associated Carbonic Anhydrase from Rat Lung. J. Biol. Chem. 267: 3308-3311.
Bonapace, G., Waheed, A., Shah, G. N., and Sly, W. S. (2004) Chemical Chaperones Protect from Effects of Apoptosis Inducing Mutation in Carbonic Anhydrase IV Identified in Retinitis Pigmentosa 17. Proc. Natl. Acad. Sci. USA 101, 12300-12305.
Bostick, D. T. and Hercules, D. M. (1975) Quantitative Determination of Blood Glucose Using Enzyme Induced Chemiluminescence of Luminol. Anal. Chem. 47, 447-452.
Zhu, X. L. and Sly, W. S. (1990) Carbonic Anhydrase IV from Human Lung. Purification, Characterization, and Comparison with Membrane Carbonic Anhydrase from Human Kidney. J. Biol. Chem. 265, 8795-8801.
White, B. H., and L. K. Kaczmarek. Identification of a Vesicular Pool of Calcium Channels in the Bag Cell Neurons of Aplysia Californica. J. Neurosci. 17, 1582-1595, 1997.